?-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) initiates the formation of ?-amyloid (A?) that has a critical role in Alzheimer's disease (AD). BACE1 is a prime AD therapeutic target and small molecule BACE1 inhibitors are in clinical trials. However, the safety and efficacy of BACE1 inhibitors have been challenged, because several BACE1 inhibitor trials have been terminated due to toxicity or lack of efficacy. BACE1 inhibitors may cause side effects, because BACE1 has many substrates with functions in neurons. The recent trial terminations suggest AD patients with dementia may be too late for clinical benefit from BACE1 inhibitors. These drugs may achieve greater efficacy at an earlier AD stage in presymptomatic individuals, so BACE1 inhibitor prevention trials are being planned. However, three key questions regarding BACE1 inhibitors for AD prevention remain unanswered. First, what level of long-term BACE1 inhibition is safe? Second, will a safe level of BACE1 inhibition slow AD pathology? Third, what A? load is too high for efficacy of BACE1 inhibition? To answer these questions, we propose three Specific Aims. In Aim 1, using adult BACE1 and BACE2 conditional knockout (cKO) mice and treatment with the BACE1 inhibitor verubecestat we will determine the thresholds of BACE1 inhibition for on- and off-target toxicities, associated substrates, and attempt rescue of spine density and hippocampal mossy fiber defects via AAV-mediated expression of soluble Sezure-6 and the Close Homolog of L1 BACE1-cleaved C-terminal fragment, respectively, two important BACE1 substrates. Comparison of genetic and pharmacologic experiments will allow us to differentiate on- vs. off-target effects of BACE1 inhibition. In Aim 2, using amyloid plaque time-stamp analysis and other methods we will study the PDAPP amyloid mouse model crossed to adult BACE1 cKO mice or treated with verubecestat to determine a safe level of BACE1 inhibition that also slows the progression of A? pathology. In Aim 3, we will perform BACE1 cKO and verubecestat experiments on PDAPP mice with different A? loads and analyze A? and pathologic phospho-tau markers by biochemistry and histology. This Aim will identify the A? load beyond which BACE1 inhibition may have little effect on tau pathology and AD progression. Together, we anticipate that this R01 to generate translational information that will be valuable for BACE1 inhibitor clinical trials for the prevention of AD.